The association of congenital heart block (CHB) with autoantibodies to SSA/Ro and SSB/La approaches the predictable. Indeed, this model of passively acquired autoimmunity offers an exceptional opportunity to examine the effector arm of immunity and define the pathogenicity of an autoantibody in mediating injury. With previous funding we characterized target antigens, examined effects of Abs on ion channels, developed techniques to culture fetal cardiac myocytes and fibroblasts, identified in vitro and in vivo apoptosis as a means of translocating Ro/La to the surface where they are accessible to maternal Abs, and initiated studies to establish a murine model. This renewal focuses on what we consider to be the most compelling mechanism to explain the low frequency and recurrence of CHB, accessibility of intracellular antigens, and the remarkable endpoint of scarring (so rare in a fetus). Accordingly, the research plan addresses the inflammatory/fibrosis cascade whereby anti-Ro/La-opsonized apoptotic cardiocytes are ingested by macrophages leading to release of inflammatory mediators and sustained fibroblast transdifferentiation. In Aim I, the effect of opsonized vs. nonopsonized apoptotic cardiocytes on macrophage phenotype and secretion will be determined. The evaluation includes: contribution of Ro/La antibodies compared to autoantibodies not known to be related to CHB; requirement and mode of induction of apoptosis; source of the macrophage. Readouts include macrophage activation markers and release of growth factors and cytokines (gene array and real-time PCR). Recognition and phagocytosis of opsonized apoptotic cardiocytes by macrophages should be distinct from apoptosis and physiologic remodeling. Aim 2 will address whether the inflammatory cascade initiated by anti-Ro/La antibodies induces a phenotypic change in the cardiac fibroblast and loss of signals that discontinue the healing process. The effect of "opsonized" supernatants on readouts relating to fibrosis will be evaluated: immunostain for transdifferentiation; functional assays such as wound contracture; GTPase intracellular signalling; and array, to identify myofibroblast specific gene products. To address inherent differences in fibroblast reactivity as a function of age or tissue location, these parameters will be evaluated in fibroblasts isolated from the fetal heart and other tissues: skin, liver, kidney, lung and adult heart. Aim 3 focuses on development of a routine model of high penetrance-CHB by exploiting candidate maternal and fetal factors to provide proof of concept. Transplacental passage of anti-Ro/La will be the initial requirement (i.e., necessary maternal factor), accomplished by active and passive immunization. A unique transgenic model expressing human 52beta in the heart (increased antigen accessibility) will be evaluated. Transgenic/knockout models will facilitate the contribution of exaggerated fetal factors involving apoptosis (increase antigen accessibility), and cytokine and growth factors (promote dysregulated wound healing). Outcome measures include: EKG abnormalities; immunohistology to evaluate apoptosis, macrophages, myofibroblasts, and scarring. By developing a mouse model with sufficient disease, pathology can be defined and subsequent therapeutic strategies addressed. The experiments embodied in this application should set precedent for defining the role of autoantibodies in other targets of injury in which sequelae are likely to involve an imbalance between wound healing and scarring.